Human pluripotent stem cells, such as human ES cells and human iPS cells, are receiving worldwide attention for their potential application to regenerative medicine. The requirement for the application of human pluripotent stem cells to regenerative medicine is to develop culture techniques for culturing and propagating such stem cells in a safe and stable manner. In particular, a pressing issue is the development of a method for stably culturing such stem cells under conditions in which no feeder cells are used (feeder-free) and no xenogeneic components are contained in the culture system (xeno-free).
Various culture matrices which enable cell culture under feeder-free and xeno-free conditions have so far been developed, and are exemplified by vitronectin, laminin α5β1γ1 (hereinafter referred to as “laminin 511”), laminin α5β2γ1 (hereinafter referred to as “laminin 521”), etc. In particular, a recombinant laminin 511E8 fragment (hereinafter referred to as “laminin 511E8”), which contains only the integrin binding site of laminin 511, was shown by the present inventors to have very strong adhesive activity for human pluripotent stem cells and be superior to any other culture matrix developed so far (see Patent Literature 1 and Non Patent Literature 1). With the use of laminin 511E8 as a culture matrix, all the culture steps including establishment, expansion culture and directed differentiation of human iPS cells can be consistently carried out under feeder-free conditions (Non Patent Literature 2).
For cell culture using laminin 511E8 as a matrix, the culture surface of a culture vessel needs coating with laminin 511E8. However, since laminin 511E8 and many other culture matrices which enable cell culture under feeder-free conditions are prepared by gene recombination techniques, the cost for preparing such recombinant proteins used for coating culture vessels is heavy economic burden in human pluripotent stem cell culture. The coating concentration of laminin 511E8 used for culture of human ES cells or human iPS cells is usually 0.25 μg/cm2 to 1.0 μg/cm2, which corresponds to 2.4 μg to 9.6 μg of laminin 511E8 per dish in the case of coating a standard 35-mm-diameter culture dish (usable surface area: 9.6 cm2), for example. Laminin 511E8 is a heterotrimer consisting of C-terminal regions of laminin α5, β1 and γ1 chains and contains many disulfide bonds in the molecule. Due to the disulfide bonds, a recombinant laminin 511E8 is difficult to produce using prokaryotic (e.g., E. coli) expression systems, and therefore, the use of animal or insect cell expression systems, albeit costly, is necessary for the production of a recombinant laminin 511E8. For popularization of the use of laminin 511E8 as a feeder-free culture matrix for human pluripotent stem cells, the production cost of laminin 511E8 needs to be reduced by improvement of the production method, and furthermore, the coating concentration of laminin 511E8 suitable for human pluripotent stem cell culture needs to be reduced by enhancement of the activity of laminin 511E8. For the success of such attempts to curb the cost for coating culture vessels, the development of a novel technique for enhancing the activity of laminin 511E8 is strongly required.
In addition, in order to popularize the culture method using laminin 511E8 domestically and abroad, there is need for the development of products which enable even an unskilled person to easily accomplish a coating operation with little variation in the coating concentration. Currently, a freeze-dried product of laminin 511E8 for coating culture vessels is commercially available (trade name: iMatrix-511, manufactured by Nippi, Inc.). In a usual procedure for use as a culture matrix, this freeze-dried laminin 511E8 product is dissolved at a concentration of 200 to 1000 μg/mL to prepare a laminin 511E8 stock solution, and this stock solution is aliquoted and kept frozen until use. For coating a culture vessel, the frozen laminin 511E8 stock solution is thawed and diluted to a desired coating concentration in PBS or the like, and the diluted solution is applied on the culture surface of the culture vessel. In this procedure, there is a risk of human errors occurring at the steps of dissolution of the freeze-dried product and dilution of the stock solution, and therefore, it is difficult to completely prevent the variation in the coating concentration among separate coating operations. If a laminin 511E8 solution previously diluted to a desired coating concentration can be used in every coating operation, even an inexperienced operator will be able to carry out a stable coating operation and thus the variation in the coating concentration will be easily prevented. In addition, the steps of thawing and diluting the laminin 511E8 stock solution will not be necessary, and thus, operation time will be significantly reduced. For these reasons, there is need for the development of a laminin 511E8 solution that does not need to be prepared immediately before use and can be stably stored without loss of the activity of laminin 511E8 for a long period of time.
The prior art disclosed in Patent Literature 2 is analogous to the present invention and is a method for enhancing an activity of laminin 511 for cells in a method for cell culture under conditions in which full-length laminin 511 has been immobilized. Specifically, Patent Literature 2 discloses “a method for enhancing an activity of laminin 511 for cells, comprising culturing mammalian cells under conditions in which laminin 511 and another polypeptide and/or peptide are immobilized, the polypeptide and/or peptide being selected from the group consisting of serum; blood proteins other than extracellular matrix proteins, such as serum albumin, prealbumin, immunoglobulin, α-globulin, β-globulin, α-1-antitrypsin (α1-AT), haptoglobin (Hp), α-2-macroglobulin (α2-M), α-fetoprotein (AFP), transferrin, retinol-binding proteins (RBPs) and adiponectin; gelatin; proteins belonging to the tumor necrosis factor (TNF) family; and peptone” (claim 7). However, the invention disclosed in Patent Literature 2 is an invention relating to a full-length laminin 511, and is completely different from the present invention in that a higher concentration of the polypeptide used in combination with the full-length laminin 511 is less effective for the enhancement of the activity.